Polynucleotides encoding tRNA methyl transferases from Streptococcus pneumoniae

ABSTRACT

The invention provides tRNA methyl transferase (trmD) polypeptides and polynucleotides encoding trmD polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing trmD polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationNo. 60/107,691 filed Nov. 9, 1998.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the trmD (tRNA methyltransferases) family, as well as their variants, herein referred to as"trmD," "trmD polynucleotide(s)," and "trmD polypeptide(s)" as the casemay be.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains that are resistantto some or all of the standard antibiotics. This phenomenon has createdan unmet medical need and demand for new anti-microbial agents,vaccines, drug screening methods, and diagnostic tests for thisorganism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces "functional genomics," that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on "positional cloning" and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the trmD embodiments of the invention, that have a present benefitof, among other things, being useful to screen compounds forantimicrobial activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and characterization of such factors andtheir antagonists and agonists to find ways to prevent, ameliorate orcorrect such infection, dysfunction and disease.

SUMMARY OF THE INVENTION

The present invention relates to trmD, in particular trmD polypeptidesand trmD polynucleotides, recombinant materials and methods for theirproduction. In another aspect, the invention relates to methods forusing such polypeptides and polynucleotides, including treatment ofmicrobial diseases, amongst others. In a further aspect, the inventionrelates to methods for identifying agonists and antagonists using thematerials provided by the invention, and for treating microbialinfections and conditions associated with such infections with theidentified agonist or antagonist compounds. In a still further aspect,the invention relates to diagnostic assays for detecting diseasesassociated with microbial infections and conditions associated with suchinfections, such as assays for detecting trmD expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to trmD polypeptides and polynucleotides asdescribed in greater detail below. In particular, the invention relatesto polypeptides and polynucleotides of a trmD of Streptococcuspneumoniae, that is related by amino acid sequence homology to B.subtilis trmD polypeptide. The invention relates especially to trmDhaving a nucleotide and amino acid sequences set out in Table 1 as SEQID NO:1 and SEQ ID NO:2 respectively. Note that sequences recited in thesequence Listing below as "DNA" represent an exemplification of theinvention, since those of ordinary skill will recognize that suchsequences can be usefully employed in polynucleotides in general,including ribopolynucleotides.

                                      TABLE 1                                     __________________________________________________________________________    trmD Polynucleptide and Polypeptide Sequences                                 __________________________________________________________________________    (A) Streptococcus pneumoniae trmD polynucleptide sequence [SEQ ID NO:1].                                                   5                                                                            '-                                  atgaagattgatattttaaccctctttccagagatgttttctccactggagcactcaatcgttggaaaggct      cgagaaaa                                                                       - agggctcttggatatccagtatcataattttcgagaaaatgctgaaaaggcccgtcatgtagatgatga                                                gcc                                 ctacggag                                                                       - gcggtcagggcatgttgctcagagcacaacctattttcaattcctttgatgctattgaaaagaaaaatc                                                cgc                                 gcgttatt                                                                       - ctcctcgatcctgctggaaagcagtttgatcaggcttatgctgaagatttggctcaagaggaagagcta                                                atc                                 tttatctg                                                                       - tgggcactatgagggttatgatgagcgcattaagaccttggtaacagatgagatttccctaggcgacta                                                tgt                                 cctcactg                                                                       - gtggagaattggcagctatgaccatgattgatgctacagttcgcctgattccagaagtgattggcaagg                                                agt                                 ctagccac                                                                       - caagatgatagtttttcttcaggtcttttagaatatcctcagtacacacgtccctatgattatcgaggc                                                atg                                 gtcgtgcc                                                                       - agatgtattgatgagtggccaccatgaaaagattcgtcagtggcgattgtacgagagtttaaagaaaac                                                cta                                 cgagcgca                                                                       - gaccagatttacttgaacattatcaactgacagtagaagaagaaaaaatgctggcagaaatcaaagaaa                                                aca                                 aagaataa                                                                     3'                                                                              - (B) Streptococcus pneumoniae trmD polypeptide sequence deduced from                                                  a polynucleotide                    sequence in this table [SEQ ID NO:2].                                         NH.sub.2 -                                                                    MKIDILTLFPEMFSPLEHSIVGKAREKGLLDIQYHNFRENAEKARHVDDEPYGGGQGMLL                  RAQPIFNSFDAIEKKNPRVI                                                           - LLDPAGKQFDQAYAEDLAQEEELIFICGHYEGYDERIKTLVTDEISLGDYVLTGGELAAM                                                          TMIDATVRLIPEVIGKESSH                                                           - QDDSFSSGLLEYPQYTRPYDYRGMVV                                                PDVLMSGHHEKIRQWRLYESLKKTYERRPD                                                LLEH                                YQLTVEEEKMLAEIKENKE                                                            - --COOH                                                                   __________________________________________________________________________

Deposited materials

A deposit comprising a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit.

On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E.coli was similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae strain deposit is referred to hereinas "the deposited strain" or as "the DNA of the deposited strain."

The deposited strain comprises a full length trmD gene. The sequence ofthe polynucleotides comprised in the deposited strain, as well as theamino acid sequence of any polypeptide encoded thereby, are controllingin the event of any conflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is comprisedin the deposited strain. Further provided by the invention are trmDpolynucleotide sequences in the deposited strain, such as DNA and RNA,and amino acid sequences encoded thereby. Also provided by the inventionare trmD polypeptide and polynucleotide sequences isolated from thedeposited strain.

Polypeptides

TrmD polypeptide of the invention is substantially phylogeneticallyrelated to other proteins of the trmD (tRNA methyl transferases) family.

In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as "trmD" and "trmDpolypeptides" as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

Among the particularly preferred embodiments of the invention arevariants of trmD polypeptide encoded by naturally occurring alleles of atrmD gene.

The present invention further provides for an isolated polypeptide that:(a) comprises or consists of an amino acid sequence that has at least95% identity, most preferably at least 97-99% or exact identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2; (b) a polypeptideencoded by an isolated polynucleotide comprising or consisting of apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1; (c) a polypeptide encoded by an isolatedpolynucleotide comprising or consisting of a polynucleotide sequenceencoding a polypeptide that has at least 95% identity, even morepreferably at least 97-99% or exact identity, to the amino acid sequenceof SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that has a biological activity oftrmD, and also those that have at least 95% identity to a polypeptide ofTable 1 [SEQ ID NO:2] and also include portions of such polypeptideswith such portion of the polypeptide generally comprising at least 30amino acids and more preferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStreptococcus pneumoniae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with trmD polypeptides, fragmentsmay be "free-standing," or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Streptococcus pneumoniae, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Polynucleotides

It is an object of the invention to provide polynucleotides that encodetrmD polypeptides, particularly polynucleotides that encode apolypeptide herein designated trmD.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding trmD polypeptides comprisinga sequence set out in Table 1 [SEQ ID NO:1] that includes a full lengthgene, or a variant thereof. The Applicants believe that this full lengthgene is essential to the growth and/or survival of an organism thatpossesses it, such as Streptococcus pneumoniae.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing trmD polypeptides andpolynucleotides, particularly Streptococcus pneumoniae trmD polypeptidesand polynucleotides, including, for example, unprocessed RNAs, ribozymeRNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Further embodiments ofthe invention include biologically, diagnostically, prophylactically,clinically or therapeutically useful polynucleotides and polypeptides,and variants thereof, and compositions comprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a trmD polypeptidehaving a deduced amino acid sequence of Table 1 [SEQ ID NO:2] andpolynucleotides closely related thereto and variants thereof

In another particularly preferred embodiment of the invention there is atrmD polypeptide from Streptococcus pneumoniae comprising or consistingof an amino acid sequence of Table 1 [SEQ ID NO:2], or a variantthereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1], a polynucleotide of the inventionencoding trmD polypeptide may be obtained using standard cloning andscreening methods, such as those for cloning and sequencing chromosomalDNA fragments from bacteria using Streptococcus pneumoniae 0100993 cellsas starting material, followed by obtaining a full length clone. Forexample, to obtain a polynucleotide sequence of the invention, such as apolynucleotide sequence given in Table 1 [SEQ ID NO:1], typically alibrary of clones of chromosomal DNA of Streptococcus pneumoniae 0100993in E. coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, New York (1989). (see in particular ScreeningBy Hybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in Table 1 [SEQ ID NO:1] was discovered in a DNAlibrary derived from Streptococcus pneumoniae 0100993.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1] contains anopen reading frame encoding a protein having about the number of aminoacid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 1 and the stopcodon that begins at nucleotide number 718 of SEQ ID NO:1, encodes thepolypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence that has at least 95% identity, even more preferably at least97, still more preferably at least 99%, yet still more preferably atleast 99.5% or exact identity to SEQ ID NO:1 over the entire length ofSEQ ID NO:1, or the entire length of that portion of SEQ ID NO:1 whichencodes SEQ ID NO:2; (b) a polynucleotide sequence encoding apolypeptide that has at least 95% identity, even more preferably atleast 97, still more preferably at least 99%, yet still more preferablyat least 99.5% or 100% exact, to the amino acid sequence of SEQ ID NO:2,over the entire length of SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5' and 3' sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 718 set forth in SEQ ID NO:1 ofTable 1, both of that encode a trmD polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

    X--(R.sub.1).sub.m --(R.sub.2)--(R.sub.3).sub.n --Y

wherein, at the 5' end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3' end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5' end nucleic acid residue is at the left,bound to R₁, and its 3' end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Streptococcus pneumoniae, however, it may preferably be obtainedfrom other organisms of the same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms of thesame taxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae trmDhaving an amino acid sequence set out in Table 1 [SEQ ID NO:2]. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may comprise coding and/or non-codingsequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingtrmD variants, that have the amino acid sequence of trmD polypeptide ofTable 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, modified, deleted and/oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, that do not alter the propertiesand activities of trmD polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO:1.

Further preferred embodiments of the invention are polynucleotides thatare at least 95%, 97% or 99.5% identical over their entire length to apolynucleotide encoding trmD polypeptide having an amino acid sequenceset out in Table 1 [SEQ ID NO:2], and polynucleotides that arecomplementary to such polynucleotides. Most highly preferred arepolynucleotides that comprise a region that is at least 95% areespecially preferred. Furthermore, those with at least 97% are highlypreferred among those with at least 95%, and among these those with atleast 98% and at least 99% are particularly highly preferred, with atleast 99.5% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to trmD polynucleotide sequences, such as thosepolynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms "stringent conditions" and "stringent hybridization conditions"mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 under stringent hybridization conditions with a probe havingthe sequence of said polynucleotide sequence set forth in SEQ ID NO:1 ora fragment thereof; and isolating said polynucleotide sequence.Fragments useful for obtaining such a polynucleotide include, forexample, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding trmD and to isolate cDNAand genomic clones of other genes that have a high identity,particularly high sequence identity, to a trmD gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have lee than 30 nucleotide residues or basepairs.

A coding region of a trmD gene may be isolated by screening using a DNAsequence provided in Table 1 [SEQ ID NO:1] to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an`adaptor` sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the "missing" 5' end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using"nested" primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3' in the adaptor sequence and a gene specific primer thatanneals further 5' in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5' primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to a mature polypeptide (when amature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for activity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO:1 readily provides contiguous fragments of SEQ ID NO:2sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextan mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fungal cells, such as cells of a yeast Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of trmD polynucleotides andpolypeptides of the invention for use as diagnostic reagents. Detectionof trmD polynucleotides and/or polypeptides in a eukaryote, particularlya mammal, and especially a human, will provide a diagnostic method fordiagnosis of disease, staging of disease or response of an infectiousorganism to drugs. Eukaryotes, particularly mammals, and especiallyhumans, particularly those infected or suspected to be infected with anorganism comprising the trmD gene or protein, may be detected at thenucleic acid or amino acid level by a variety of well known techniquesas well as by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled trmD polynucleotide sequences.Perfectly or significantly matched sequences can be distinguished fromimperfectly or more significantly mismatched duplexes by DNase or RNasedigestion, for DNA or RNA respectively, or by detecting differences inmelting temperatures or renaturation kinetics. Polynucleotide sequencedifferences may also be detected by alterations in the electrophoreticmobility of polynucleotide fragments in gels as compared to a referencesequence. This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401 (1985).

In another embodiment, an array of oligonucleotides probes comprisingtrmD nucleotide sequence or fragments thereof can be constructed toconduct efficient screening of, for example, genetic mutations,serotype, taxonomic classification or identification. Array technologymethods are well known and have general applicability and can be used toaddress a variety of questions in molecular genetics including geneexpression, genetic linkage, and genetic variability (see, for example,Chee et al., Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit that comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding trmD polypeptide can be used to identity andanalyze mutations. The invention further provides these primers with 1,2, 3 or 4 nucleotides removed from the 5' and/or the 3' end. Theseprimers may be used for, among other things, amplifying trmD DNA and/orRNA isolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1].Increased or decreased expression of a trmD polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of trmD polypeptide compared to normal controltissue samples may be used to detect the presence of an infection, forexample. Assay techniques that can be used to determine levels of a trmDpolypeptide, in a sample derived from a host, such as a bodily material,are well-known to those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis,antibody sandwich assays, antibody detection and ELISA assays.

Antagonists and Agonists--Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g., inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, oftrmD polypeptides and polynucleotides; or may be structural orfunctional mimetics thereof (see Coligan et al., Current Protocols inImmunology 1(2):Chapter 5 (1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists, in the absence of an agonist or antagonist, by testing whetherthe candidate compound results in inhibition of activation of thepolypeptide or polynucleotide, as the case may be. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring trmD polypeptideand/or polynucleotide activity in the mixture, and comparing the trmDpolypeptide and/or polynucleotide activity of the mixture to a standard.Fusion proteins, such as those made from Fc portion and trmDpolypeptide, as herein described, can also be used for high-throughputscreening assays to identify antagonists of the polypeptide of thepresent invention, as well as of phylogenetically and and/orfunctionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose that enhance (agonist) or block (antagonist) the action of trmDpolypeptides or polynucleotides, particularly those compounds that arebacteristatic and/or bactericidal. The method of screening may involvehigh-throughput techniques. For example, to screen for agonists orantagonists, a synthetic reaction mix, a cellular compartment, such as amembrane, cell envelope or cell wall, or a preparation of any thereof,comprising trmD polypeptide and a labeled substrate or ligand of suchpolypeptide is incubated in the absence or the presence of a candidatemolecule that may be a trmD agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the trmD polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of trmD polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocalorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in trmD polynucleotide or polypeptideactivity, and binding assays known in the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance, ¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by trmD polypeptide associating with anothertrmD polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of trmD polypeptide dimers,trimers, tetramers or higher order structures, or structures formed bytrmD polypeptide bound to another polypeptide. TrmD polypeptide can belabeled with both a donor and acceptor fluorophore. Upon mixing of thetwo labeled species and excitation of the donor fluorophore,fluorescence energy transfer can be detected by observing fluorescenceof the acceptor. Compounds that block dimerization will inhibitfluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on trmD polypeptide self-association as well as an associationof trmD polypeptide and another polypeptide or small molecule. TrmDpolypeptide can be coupled to a sensor chip at low site density suchthat covalently bound molecules will be monomeric. Solution protein canthen passed over the trmD polypeptide -coated surface and specificbinding can be detected in real-time by monitoring the change inresonance angle caused by a change in local refractive index. Thistechnique can be used to characterize the effect of small molecules onkinetic rates and equilibrium binding constants for trmD polypeptideself-association as well as an association of trmD polypeptide andanother polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of trmD polypeptide with another trmDpolypeptide or a different polypeptide. TrmD polypeptide can be coupledto a scintillation-filled bead. Addition of radio-labeled trmDpolypeptide results in binding where the radioactive source molecule isin close proximity to the scintillation fluid. Thus, signal is emittedupon trmD polypeptide binding and compounds that prevent trmDpolypeptide self-association or an association of trmD polypeptide andanother polypeptide or small molecule will diminish signal.

In other embodiments of the invention there are provided methods foridentifying compounds that bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for trmD agonists is a competitive assaythat combines trmD and a potential agonist with trmD-binding molecules,recombinant trmD binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. TrmD can be labeled, such as byradioactivity or a colorimetric compound, such that the number of trmDmolecules bound to a binding molecule or converted to product can bedetermined accurately to assess the effectiveness of the potentialantagonist.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist or antagonist of thepolypeptide and/or polynucleotide, by: (a) determining in the firstinstance the three-dimensional structure of the polypeptide and/orpolynucleotide, or complexes thereof, (b) deducing the three-dimensionalstructure for the likely reactive site(s), binding site(s) or motif(s)of an agonist or antagonist; (c) synthesizing candidate compounds thatare predicted to bind to or react with the deduced binding site(s),reactive site(s), and/or motif(s); and (d) testing whether the candidatecompounds are indeed agonists or antagonists.

It will be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of trmD polypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the trmDpolypeptide and/or polypeptide.

In still another approach, expression of the gene encoding endogenoustrmD polypeptide can be inhibited using expression blocking techniques.This blocking may be targeted against any step in gene expression, butis preferably targeted against transcription and/or translation. Anexamples of a known technique of this sort involve the use of antisensesequences, either internally generated or separately administered (see,for example, O'Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides thatform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial trmD proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided trmD agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter Pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists oftrmD polypeptides and/or polynucleotides) found using screens providedby the invention, or known in the art, particularly narrow-spectrumantibiotics, should be useful in the treatment of H. pylori infection.Such treatment should decrease the advent of H. pylori-induced cancers,such as gastrointestinal carcinoma. Such treatment should also prevent,inhibit and/or cure gastric ulcers and gastritis.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

"Disease(s)" means any disease caused by or related to infection by abacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

"Host cell(s)" is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,"identity" also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. "Identity"can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97, 99.5 or 100% identity to the reference sequence of SEQ ID NO:1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO:1 or may include up to a certain integer number ofnucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5' or 3' terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1, or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.95 for 95%, 0.97 for97%, 0.995 for 99.5% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

"Individual(s)" means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is "isolated" even if it is still present in saidorganism, which organism may be living or non-living.

"Organism(s)" means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfingens, Clostridiumtetani, Clostridium botulinum, Treponema pallidum, Rickettsia rickettsiiand Chlamydia trachomitis, (ii) an archaeon, including but not limitedto Archaebacter, and (iii) a unicellular or filamentous eukaryote,including but not limited to, a protozoan, a fungus, a member of thegenus Saccharomyces, Kluveromyces, or Candida, and a member of thespecies Saccharomyces ceriviseae, Kluveromyces lactis, or Candidaalbicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS--STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Recombinant expression system(s)" refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and Ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones comprising overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO:1. Librariesmay be prepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 trmD Characterization

The S. pneumoniae trmD gene is expressed during infection in arespiratory tract infection model

The determination of expression during infection of a gene fromStreptococcus pneumoniae Excised lungs from a 48 hour respiratory tractinfection of Streptococcus pneumoniae 0100993 in the mouse isefficiently disrupted and processed in the presence of chaotropic agentsand RNAase inhibitor to provide a mixture of animal and bacterial RNA.The optimal conditions for disruption and processing to give stablepreparations and high yields of bacterial RNA are followed by the use ofhybridisation to a radiolabelled oligonucleotide specific toStreptococcus pneumoniae 16S RNA on Northern blots. The RNAase free,DNAase free, DNA and protein free preparations of RNA obtained aresuitable for Reverse Transcription PCR (RT-PCR) using unique primerpairs designed from the sequence of each gene of Streptococcuspneumoniae 0100993.

a) Isolation of tissue infected with Streptococcus pneumoniae 0100993from a mouse animal model of infection (lungs)

Streptococcus pneumoniae 0100993 is seeded onto TSA (Tryptic Soy Agar,BBL) plates containing 5% horse blood and allowed to grow overnight at37° C. in a CO2 incubator. Bacterial growth is scraped into 5 ml ofphosphate-buffered saline (PBS) and adjusted to an A600˜0.6 (4×106/ml).Mice (male CBA/J-1 mice, approximately 20 g) were anaesthetized withisoflurane and 50 microliters of the prepared bacterial inoculum isdelivered by intranasal instillation. Animals are allowed to recover andobserved twice daily for signs of moribundancy. Forty-eight hours afterinfection the animals are euthanized by carbon dioxide overdose andtheir torsos swabbed with ethanol and then RNAZap. The torso is thenopened, and the lungs are aseptically removed. Half of each pair oflungs is placed in a cryovial and immediately frozen in liquid nitrogen;the other half is used for bacterial enumeration after homogenization ofthe tissue in 1 ml of PBS.

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infectedtissue samples

Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from-80° C. storage into a dry ice ethanol bath. In a microbiological safetycabinet the samples are disrupted up to eight at a time while theremaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroforn/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at-80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at -80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at -20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1×TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1×MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a32P-labelled oligonucletide probe, of sequence 5'AACTGAGACTGGCTTTAAGAGATTA 3' [SEQ ID NO:3], specific to 16S rRNA ofStreptococcus pneumoniae. The size of the hybridising band is comparedto that of control RNA isolated from in vitro grown Streptococcuspneumoniae 0100993 in the Northern blot. Correct sized bacterial 16SrRNA bands can be detected in total RNA samples which show degradationof the mammalian RNA when visualised on TBE gels.

c) The removal of DNA from Streptococcus pneumoniae-derived RNA

DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol.

DNAase treated RNA was resuspended in 100 microliters of DEPC treatedwater with the addition of Rnasin as described before.

d) The preparation of cDNA from RNA samples derived from infected tissue

3 microgram samples of DNAase treated RNA are reverse transcribed usinga SuperScript Preamplification System for First Strand cDNA Synthesiskit (Gibco BRL, Life Technologies) according to the manufacturersinstructions. 150 nanogram of random hexamers is used to prime eachreaction. Controls without the addition of SuperScriptII reversetranscriptase are also run. Both +/-RT samples are treated with RNaseHbefore proceeding to the PCR reaction

e) The use of PCR to determine the presence of a bacterial cDNA species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 43 microliters PCR Master Mix (Advanced BiotechnologiesLtd.); 1 microliter PCR primers (optimally 18-25 basepairs in length anddesigned to possess similar annealing temperatures), each primer at 10mM initial concentration; and 5 microliters cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at 94°C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then a holdtemperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microliter aliquots are then run out on 1%1×TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5'endwith a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScan™ software as supplied by Perkin Elmer).

RT/PCR controls may include +/-reverse transcriptase reactions, 16S rRNAprimers or DNA specific primer pairs designed to produce PCR productsfrom non-transcribed Streptococcus pneumoniae 0100993 genomic sequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStreptococcus pneumoniae 0100993 total DNA. PCR reactions are set up andrun as described above using approx. 1 microgram of DNA in place of thecDNA.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1. Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR, and 2. Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls.

Example 3

The trmD gene is essential for S. pneumoniae in vitro growth.

Demonstration of gene essentiality to bacterial viability

An allelic replacement cassette was generated using PCR technology. Thecassette consisted of a pair of 500 bp chromosomal DNA fragmentsflanking an erythromycin resistance gene. The chromosomal DNA sequencesare the 500 bp preceding and following the DNA sequence encoding thetrmD gene contained in Seq. ID NO.1

The allelic replacement cassette was introduced into S. pneumoniae R₆ bytransformation. Competent cells were prepared according to publishedprotocols. DNA was introduced into the cells by incubation of ngquantities of allelic replacement cassette with 10⁶ cells at 30° C. for30 minutes. The cells were transferred to 37° C. for 90 minutes to allowexpression of the erythromycin resistance gene. Cells were plated inagar containing lug erythromycin per ml. Following incubation at 37° C.for 36 hours, colonies are picked and grown overnight in Todd-Hewittbroth supplemented with 0.5% yeast extract. Typically 1000 transformantscontaining the appropriate allelic replacement are obtained. If notransformants are obtained in three separate transformation experimentsas was the case for this gene trmD, then the gene is considered as beingessential in vitro.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 3                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 720                                                             <212> TYPE: DNA                                                               <213> ORGANISM: Streptococcus pneumoniae                                       - - <400> SEQUENCE: 1                                                         - - atgaagattg atattttaac cctctttcca gagatgtttt ctccactgga gc -            #actcaatc     60                                                                 - - gttggaaagg ctcgagaaaa agggctcttg gatatccagt atcataattt tc -            #gagaaaat    120                                                                 - - gctgaaaagg cccgtcatgt agatgatgag ccctacggag gcggtcaggg ca -            #tgttgctc    180                                                                 - - agagcacaac ctattttcaa ttcctttgat gctattgaaa agaaaaatcc gc -            #gcgttatt    240                                                                 - - ctcctcgatc ctgctggaaa gcagtttgat caggcttatg ctgaagattt gg -            #ctcaagag    300                                                                 - - gaagagctaa tctttatctg tgggcactat gagggttatg atgagcgcat ta -            #agaccttg    360                                                                 - - gtaacagatg agatttccct aggcgactat gtcctcactg gtggagaatt gg -            #cagctatg    420                                                                 - - accatgattg atgctacagt tcgcctgatt ccagaagtga ttggcaagga gt -            #ctagccac    480                                                                 - - caagatgata gtttttcttc aggtctttta gaatatcctc agtacacacg tc -            #cctatgat    540                                                                 - - tatcgaggca tggtcgtgcc agatgtattg atgagtggcc accatgaaaa ga -            #ttcgtcag    600                                                                 - - tggcgattgt acgagagttt aaagaaaacc tacgagcgca gaccagattt ac -            #ttgaacat    660                                                                 - - tatcaactga cagtagaaga agaaaaaatg ctggcagaaa tcaaagaaaa ca -            #aagaataa    720                                                                 - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 239                                                             <212> TYPE: PRT                                                               <213> ORGANISM: Streptococcus pneumoniae                                       - - <400> SEQUENCE: 2                                                         - - Met Lys Ile Asp Ile Leu Thr Leu Phe Pro Gl - #u Met Phe Ser Pro        Leu                                                                              1               5  - #                10  - #                15              - - Glu His Ser Ile Val Gly Lys Ala Arg Glu Ly - #s Gly Leu Leu Asp Ile                  20      - #            25      - #            30                   - - Gln Tyr His Asn Phe Arg Glu Asn Ala Glu Ly - #s Ala Arg His Val Asp              35          - #        40          - #        45                       - - Asp Glu Pro Tyr Gly Gly Gly Gln Gly Met Le - #u Leu Arg Ala Gln Pro          50              - #    55              - #    60                           - - Ile Phe Asn Ser Phe Asp Ala Ile Glu Lys Ly - #s Asn Pro Arg Val Ile      65                  - #70                  - #75                  - #80        - - Leu Leu Asp Pro Ala Gly Lys Gln Phe Asp Gl - #n Ala Tyr Ala Glu Asp                      85  - #                90  - #                95               - - Leu Ala Gln Glu Glu Glu Leu Ile Phe Ile Cy - #s Gly His Tyr Glu Gly                  100      - #           105      - #           110                  - - Tyr Asp Glu Arg Ile Lys Thr Leu Val Thr As - #p Glu Ile Ser Leu Gly              115          - #       120          - #       125                      - - Asp Tyr Val Leu Thr Gly Gly Glu Leu Ala Al - #a Met Thr Met Ile Asp          130              - #   135              - #   140                          - - Ala Thr Val Arg Leu Ile Pro Glu Val Ile Gl - #y Lys Glu Ser Ser His      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Gln Asp Asp Ser Phe Ser Ser Gly Leu Leu Gl - #u Tyr Pro Gln Tyr        Thr                                                                                             165  - #               170  - #               175             - - Arg Pro Tyr Asp Tyr Arg Gly Met Val Val Pr - #o Asp Val Leu Met Ser                  180      - #           185      - #           190                  - - Gly His His Glu Lys Ile Arg Gln Trp Arg Le - #u Tyr Glu Ser Leu Lys              195          - #       200          - #       205                      - - Lys Thr Tyr Glu Arg Arg Pro Asp Leu Leu Gl - #u His Tyr Gln Leu Thr          210              - #   215              - #   220                          - - Val Glu Glu Glu Lys Met Leu Ala Glu Ile Ly - #s Glu Asn Lys Glu          225                 2 - #30                 2 - #35                            - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Streptococcus pneumoniae                                       - - <400> SEQUENCE: 3                                                         - - aactgagact ggctttaaga gatta          - #                  - #                   25                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment comprising afirst polynucleotide sequence or the full complement of the entirelength of the first polynucleotide sequence, wherein the firstpolynucleotide sequence encodes a polypeptide comprising the amino acidsequence set forth in SEQ ID NO:2.
 2. The isolated polynucleotidesegment of claim 1, wherein the isolated polynucleotide segmentcomprises the first polynucleotide sequence.
 3. A vector comprising theisolated polynucleotide segment of claim
 2. 4. An isolated host cellcomprising the vector of claim
 3. 5. A process for producing apolypeptide comprising the step of culturing the host cell of claim 4under conditions sufficient for the production of the polypeptide,wherein the polypeptide is encoded by the first polynucleotide sequence.6. The isolated polynucleotide segment of claim 1, wherein the isolatedpolynucleotide segment comprises the full complement of the entirelength of the first polynucleotide sequence.
 7. A vector comprising theisolated polynucleotide segment of claim
 6. 8. An isolated host cellcomprising the vector of claim
 7. 9. The isolated polynucleotide ofclaim 2 encoding a fusion polypeptide, wherein the first polynucleotideencodes part of the fusion polypeptide.
 10. An isolated polynucleotidesegment comprising a first polynucleotide sequence or the fullcomplement of the entire length of the first polynucleotide sequence,wherein the first polynucleotide sequence comprises SEQ ID NO:1.
 11. Theisolated polynucleotide segment of claim 10, wherein the isolatedpolynucleotide segment comprises the first polynucleotide sequence. 12.A vector comprising the isolated polynucleotide segment of claim
 11. 13.An isolated host cell comprising the vector of claim
 12. 14. A processfor producing a polypeptide comprising the step of culturing the hostcell of claim 13 under conditions sufficient for the production of thepolypeptide, wherein the polypeptide is encoded by the firstpolynucleotide sequence.
 15. The isolated polynucleotide segment ofclaim 10, wherein the isolated polynucleotide segment comprises the fullcomplement of the entire length of the first polynucleotide sequence.16. A vector comprising the isolated polynucleotide segment of claim 15.17. An isolated host cell comprising the vector of claim
 16. 18. Theisolated polynucleotide of claim 11 encoding a fusion polypeptide,wherein the first polynucleotide encodes part of the fusion polypeptide.19. An isolated polynucleotide segment comprising a first polynucleotidesequence or the full complement of the entire length of the firstpolynucleotide sequence, wherein the first polynucleotide sequenceencodes a polypeptide consisting of the amino acid sequence set forth inSEQ ID NO:2.
 20. The isolated polynucleotide segment of claim 19,wherein the isolated polynucleotide segment comprises the firstpolynucleotide sequence.
 21. A vector comprising the isolatedpolynucleotide segment of claim
 20. 22. An isolated host cell comprisingthe vector of claim
 21. 23. A process for producing a polypeptidecomprising the step of culturing the host cell of claim 22 underconditions sufficient for the production of the polypeptide, wherein thepolypeptide is encoded by the first polynucleotide sequence.
 24. Theisolated polynucleotide segment of claim 19, wherein the isolatedpolynucleotide segment comprises the full complement of the entirelength of the first polynucleotide sequence.
 25. A vector comprising theisolated polynucleotide segment of claim
 24. 26. An isolated host cellcomprising the vector of claim 25.